Serum Response Factor, an Enriched Cardiac Mesoderm Obligatory Factor, Is a Downstream Gene Target for Tbx Genes

Matthew R Barron,Narasimhaswamy S Belaguli,Shu Xiang Zhang,Mimi Trinh,Dinaker Iyer,Xanthi Merlo,John W Lough,Michael S Parmacek,Benoit G Bruneau,Robert J Schwartz

doi:10.1074/jbc.m412408200

Abstract

We tested the idea that T-box factors direct serum response factor (SRF) gene activity early in development. Analysis of SRF-LacZ "knock-in" mice showed highly restricted expression in early embryonic cardiac and skeletal muscle mesoderm and neuroectoderm. Examination of the SRF gene for regulatory regions by linking the promoter and 5'-flanking sequences, up to 5.5 kb, failed to target LacZ transgene activity to the heart and the tail pre-somitic mesenchyme. However, linkage of a minimal SRF promoter with the SRF 3'-untranslated region (UTR), inundated with multimeric T-box binding sites (TBEs), restored robust reporter gene activity to embryonic heart and tail. Finer dissection of the 3'-UTR to a small cluster of TBEs also stimulated transgene activity in the cardiac forming region and the tail, however, when the TBEs contained within these DNA sequences were mutated, preventing Tbx binding, transgene activity was lost. Tbx2, Tbx5, and the cardiac-enriched MYST family histone acetyltransferase TIP60, were observed to be mutual interactive cofactors through the TIP60 zinc finger and the T-box of the Tbx factors. In SRF-null ES cells, TIP60, Tbx2, and Tbx5 were sufficient to stimulate co-transfected SRF reporter activity, however this activity required the presence of the SRF 3'-UTR. SRF gene transactivation was blocked by two distinct TIP60 mutants, in which either the histone acetyltransferase domain was inactivated or the Zn finger-protein binding domain was excised. Our study supports the idea that SRF embryonic cardiac gene expression is dependent upon the SRF 3'-UTR enhancer, Tbx2, Tbx5, and TIP60 histone acetyltransferase activity.

Highlights

During early development, a subset of pluripotent mesodermal cells becomes increasingly committed to the cardiac muscle lineage, through the combinatorial interactions of transcription factors, which result in the expression of cardiac-specific genes
The concentration of serum response factor (SRF) in muscle precursors early in development suggests that SRF plays a specific role in muscle specification and differentiation
A Potential Candidate Role for Tbx Factors to Direct SRF Gene Activity—We reported on the appearance of Tbx2, Tbx3, and Tbx5 transcripts during early chick embryogenesis [23] and noticed that Tbx2 and Tbx5 proteins were expressed in regions of the developing heart that might be co-expressed with SRF

Summary

Introduction

A subset of pluripotent mesodermal cells becomes increasingly committed to the cardiac muscle lineage, through the combinatorial interactions of transcription factors, which result in the expression of cardiac-specific genes. Analysis of SRF null mice revealed a severe block to the activation of SRF-regulated immediate early genes, but it showed inhibited expression of the cardiac, skeletal, and smooth muscle ␣-actins. The regulatory regions of a number of muscle-specific genes, such as skeletal, cardiac (6 – 8), and smooth muscle ␣-actin [9, 10], contain serum response elements, which are required for promoter activity and depend upon SRF (see a recent review, Ref. 11). We found that the promoter sequences containing the GATA, Nkx, and SRF binding sites were insufficient to drive expression in the developing heart, skeletal and smooth muscle populations expressed SRF in these mice. Where are the SRF genetic sequences that allow for cardiac expression?

Methods

Results

Conclusion